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УДК 616. 133−009. 86:612. 73. 014HYPOXiA-iNDUCED 15-HETE ENHANCES THE CONSTRiCTiON OF iNTERNAL CAROTiD ARTERiESby down-regulating potassium channelsYanmei Zhu1, Li Chen1, Wenjuan Liu1, Weizhi Wang1, Daling Zhu2, Yulan Zhu11 Second Affiliated Hospital of Harbin Medical University, 2 College of Pharmacy of Harbin Medical University (157 Baojian Road, Nangang District, Harbin 150 081 P.R. China)Keywords: voltage-gated potassium channel, smooth muscle cells, cerebral vasoconstriction.Summary — Severe hypoxia induces the constriction of internal carotid arteries (ICA), which worsens ischemic stroke in the brain. A few metabolites are presumably involved in hypoxic vasoconstriction, however, less is known about how such molecules provoke this vasoconstriction. We have investigated the influence of 15-hy-droxyeicosatetrienoic acid (15-HETE) produced by 15-lipoxygenase (15-LOX) on vasoconstriction during hypoxia. As showed in our results, 15-LOX level increases in ICA endothelia and smooth muscles. 15-HETE enhances the tension of ICA ring in a dose-dependent manner, as well as attenuates the activity and expression of voltage-gated potassium (Kv) channels. Therefore, the down-regulation of Kv channels by 15-HETE during hypoxia weakens the repolarization of action potentials and causes a dominant influx of calcium to enhance smooth muscle tension and ICA constriction.The pathogenesis of cerebral ischemia includes thrombosis and hypoxia-induced vascular constriction, which lead to cell death [3]. The efforts to treat hypoxia-induced cerebral vasoconstriction in ischemia has been failed to achieve the primary goal. The reexamination of its underlying mechanisms is needed [10]. In addition to calcium influx, as hypoxia inhibits voltage-gated potassium (Kv) channels, the inhibition of Kv channels may be involved in hypoxic vasoconstriction through prolonging repolarization period for calcium entry [5, 8].There are four subtypes of potassium channels in vascular smooth muscle cells, Kv, ATP-sensitive K+, inward rectification and large-conductance Ca2±activated K+ [4]. Kv channels are encoded by genes of Kv1. 0-Kv9. 0, KvLQT and EAG, and some of them (Kv1. 2, Kv1.5 and Kv2.1 [7]) are sensitive to hypoxia [11]. Which subtypes of Kv channels contribute to hypoxic cerebral vasoconstriction? In addition, several endothelial agents, e.g., endothelin, prostaglandin, leukotriene and cytochrome P450 metabolites, as well as 15-hydroxyeicosatetrienoic acid (15-HETE) induce hypoxic vasoconstriction [2, 12]. Hypoxia induces the expression of vascular 15-lipoxygenase (15-LOX) and increases the sensitivity of cerebral arteries to 15-HETE [14, 15]. Does 15-HETE serve as an essential mediator between hypoxia and Kv-channel inhibition for cerebral vasoconstriction?To these questions, we examined the expression of 15-LOX by immunohistochemistry in vivo, the role of 15-HETE in hypoxic internal carotid arteries constriction by measuring its tension, the expressions of Kv2. 1/Kv1.5 by western-blot and RT-PCR, as well as the activity of Kv channels by whole-cell recording in cerebral arterial smooth muscle cells (CASMC) of rats. Our study reveals that 15-HETE induces hypoxic cerebral vasoconstriction via down-regulating Kv channels.Yulan Zhu — Department of Neurology. Tel.: 86−451−89 778 959, 86−15 134 556 239, Fax: 86−451−86 605 656, e-mail: ylz1962@yahoo. com. cnMethodsWistar rats (225±25 g) were housed in the Animal Resource Center of Harbin Medical University. The procedures are approved by Institutional Animal Care and Use Committee. Rats were randomly divided into two groups. Group one was placed in normoxic environment as control, and another was maintained in a cage where fractional inspired oxygen (FiO2) was reduced to 0. 12 as hypoxic condition [14]. Living temperature was 22±2 °C, and relative humidity was 50±10%. After nine days, rats were anesthetized by the intraperitoneal injection of 4% sodium pentobarbital (40 mg/kg), and internal carotid arteries were surgically taken for subsequent experiments.Internal carotid arteries rings were cut into 1. 0−1.5 mm in length. Each fragment was mounted onto a tungsten wire, and immersed into an oxygenized KH solution (mM: NaCl, 118- KCl, 4. 7- CaCl2, 2. 5- MgSO4, 1. 2- NaHCO3, 2. 5- KH2PO4, 1. 2- glucose, 6. 0- pH 7. 4) at 37 °C. After this equilibration for 40 min, the rings were loaded with a tension of 0.3 g. The relationships between vasoconstriction in internal carotid arteries rings and dose-response for 15-HETE were assessed in normoxic and hypoxic groups (n=8). 15-HETE (Cayman Chemical Company in USA) was added into KH solution from 10−8 M to 10−6 at 3 min intervals up to final concentrations.The immunohistochemistry of 15-LOX: Rats were anesthetized (see above). 4% parafolmadehyde in 0.1 M phosphate buffer solution (PBS) was perfused into left ventricle/ aorta until their bodies were rigidity. The brains were quickly isolated and fixed in 4% parafolmadehyde PBS for additional 24 hrs. Cortical tissues were sliced in cross section at 20m by a freezing microtome. Sections were washed by PBS for three times and stained by 15-LOX immunohistochemistry [14]. The distribution of 15-LOX (dark-brown in color) was observed under conventional optical microscope.The culture of rat CASMCs: internal carotid arteries rings were cut into small pieces, dispersed in cultural medium with 4 mg/ml papain [6] for 18 min at 37 °C, and transferred into the medium with collagenase (1 mg/ml, In-vitrogen USA) [1] for 20 min at 37 °C. The isolated vascular smooth muscle cells were transferred and stirred in DMEM solution supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin. The solution was centrifuged for 10 min to have cell pellets. The resuspended cells were distributed into a plate with 6 orifices and cultured in a humidified incubator (37 °C, 5% CO2) for 3−5 days. The purity of CASMCs in primary cultures was confirmed by specific monoclonal antibody for smooth-muscle -actin(Boehringer Mannheim). Before experiments, cell growth was stopped by adding in 0.3% FBS-DMEM for 12h. Quiescent (growth-arrested) CASMCs were divided into three groups. Group one (normoxic control) was maintained in an incubator with a 5% CO2 / 95% O2. Hypoxia group were incubated in gas mixture composed of 3% O2, 5% CO2, and 92% N2. 1 mmol/L 15-HETE was added into the third group in an incubator containing 5% CO2 and 95% O2.Electrophysiological experiments: In voltage-clamp of CASMCs, Kv channel currents were evoked by depolarization pulses, which were isolated by adding TTX and nimodipine (10 uM). An Axon-Patch 200B amplifier (Axon Instrument, Foster CA, USA) produced depolarization pulses to clamp membrane potentials to different levels and recorded outward Kv currents. Electrical signals were inputted into pClamp-9 (Axon Instrument) for data acquisition and analysis. Transient capacitance was compensated, and output bandwidth was 3 kHz. Standard pipette solution for whole-cell recording contained (mM) 150 K-gluconate, 5 NaCl, 0.4 EGTA, 4 Mg-ATP, 0.5 Tris-GTP and 4 Na-phosphocreatine, 10 HEPES (pH 7.4 adjusted by 2M KOH). The osmolarity of pipette solution was 295−305 mOsmol, and the resistance of pipettes was 8−10 MW to have good access and prevent run-down in synaptic responses.Western blotting: Primary-cultured CASMCs were gently washed thrice in cold PBS and placed in 200l lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl-sulfate, 100 ^g/ml phenylmethylsulfonyl fluoride and 30 ^l/ml aprotinin) for 30 min on ice. The lysates were sonicated, and centrifuged at 12,000 rpm for 10 min at 4 °C. Proteins in supernatant were quantified based on a method of Bradford, separated by sodium dodecyl-sulfate-PAGE and transferred to nitrocellulose membranes. The membranes were incubated in a blocking buffer (TBST solution with nonfat dry milk powder) for 1 hour at room temperature, and placed in affinity-purified rabbit polyclonal antibodies (1: 500) specific to Kv1.5 and Kv2.1 (Santa Cruz USA) overnight at 4 °C. Monoclonal antibody specific to smooth muscle P-actin was used for control. The membranes were washed and incubated with anti-rabbit horseradish peroxidase-conjugated IgG (1: 5000) for 1 hour at room temperature. Bound antibodies were detected with an enhanced chemiluminescence-detection system (Amersham). The bands corresponding to the expected size were selected on a computerized scanner, and the pixel density within each band was determined by this computer after background correction for relative quantization.RT-PCR: Sequences for rat Kv1.5 and Kv2.1 were obtained from GenBankTM database. Primers for rKv1. 5a (M27158) are sense 5'--GGGCAAGATCGTGGGTT-3'- and antisense 5 '--GGCTTAAATACTCGGTGGTG-3'- with 460 bp fragment. Primers for rKv2. 1a (X16476) are sense 5'--CACCATCGCTCTGTCACTCA-3'- and antisense 5 '--GCAGGCCCAGTTCGTTGTA-3'- with fragment size 395 bp. Primers for P-actin (BC063166) are sense 5 '--CCGTAAAGACCTCTATGCCAACA- 3'- and antisense 5 '--CGGACTCATCGTACTCCTGCT-3'- with fragment size: 230 bp.Total RNAs from primary-cultured CASMCs were extracted by Trizol and reversely transcribed by cDNA synthesis kit (Fermentas). The fidelity and specificity of sense and antisense oligonucleotides were tested with BLAST. cDNA samples were amplified in DNA thermal cycler (PerkinElmer). PCR products were electrophoresed through a 1% agarose gel. cDNA bands were visualized by GelStar gel staining (FMC BioProducts). Invariant mRNA of P-actin was used as an internal control to quantify PCR products. OD values for channel signals, measured by a Kodak electrophoresis documentation system, were normalized to OD values of P-actin signals. The ratios were expressed as arbitrary units for quantitative comparison.Statistical analysis: All values were presented as means and standard error, and calculated by using two-tailed analyses of variance (ANOVA) followed by Dunnett'-s test to examine the significance among experimental groups.Results15-HETE enhances the sensitivity of ICA to hypoxia-induced vasoconstrictionThe expression of 15-LOX in internal carotid arteries was examined during hypoxia. Two groups of rats were placed under the conditions of hypoxia and control, respectively (see Methods). Nine days after the treatments, rats'- brain tissues were isolated for immunohistochemistry. As showed in top panels of Fig. 1, the levels of 15-LOX in the layers of endothelia and smooth muscles are higher in hypoxia (1b) than control (1a), indicating that hypoxia elevates 15-LOX expression.As 15-LOX produces 15-HETE, we tested the effect of 15-HETE on the sensitivity of internal carotid arteries tension to hypoxia. Figure 1c shows the different concentrations of 15-HETE vs. the tension of internal carotid arteries rings under the conditions of control (open symbols) and hypoxia (filled). Internal carotid arteries tension is higher under hy-poxia than control in a dose-dependent manner, indicating that 15-HETE enhances the sensitivity of internal carotid arteries to hypoxia. Therefore, hypoxia may induce 15-LOX over-expression, and subsequently synthesized 15-HETE strengthens internal carotid arteries constriction.In terms of mechanisms underlying hypoxic 15-HETE vasoconstriction, we studied the involvement of Kv channels, since Kv1. 2, Kv1.5 and Kv2.1 were sensitive to hypoxia [4, 7]. If 15-HETE reduces Kv quantity and/or function, a delayed repolarization leads to dominant Ca2+ influx and enhances smooth muscle tension, i.e., hypoxic vasoconstriction. 15-HETE reduces the expression and function of Kv channelsWe examined whether 15-HETE reduced the expressions of Kv channels in smooth muscles by using western-blot and RT-PCR, respectively, in which the levels of Kv proteins and mRNAs were read out by an enhanced chemiluminescence-detection system and a computerized scanner. Cerebral arterial smooth muscle cells (CASMC) were harvested and cultured under the conditions of hy-poxia, 15-HETE and control (see Methods). Five days after the culture, CASMCs were collected in consistent quantities from three groups for the experiments.: Wr